Volatile organochlorides (VOC's) are common environmental pollutants known for their toxicity and the hazards presented when in contact with humans. VOC's include trichloroethylene, dichloroethylene, vinyl chloride, ethylene chloride and the like. Trichloroethylene (TCE) is an especially well known VOC, used for a variety of purposes such as for solvent extraction, as a degreaser in dry cleaning and the manufacture of organic chemicals, pharmaceuticals and the like. Leaky above or below underground storage tanks, spills or improper disposal of waste materials containing VOC's such as TCE can pollute aquifers, soils, ground water, waste water and the like.
A number of techniques have been developed to degrade or remove TCE and related VOC's under a number of conditions. However, they are typically quite limited in their flexibility of use and have proven to be less than effective. Many of these efforts have concentrated in the microbial degradation of TCE and VOC's. However, the efforts to date have fallen short of the desire to cost effectively and completely remediate soils and/or contaminated ground water and/or waste materials.
To date, most biological systems for degrading TCE require the use of specialized microorganisms and/or the induction of specialized genes. Gene induction is the process by which genes are activated and remain in an activated mode. Gene induction typically requires the presence of an inducing co-substrate, many of which are toxic, hazardous substances.
For example, Shields et al in "Selection of a Pseudomonas cepacia Strain Constitutive for the Degradation of Trichloroethylene", Applied and Environmental Microbiology, Vol. 58, p. 3977-3983 (December 1992), employs recombinant Pseudomonas cepacia in an attempt to degrade TCE, toluene, phenol or m-trifluoromethyl phenol. Pseudomonas cepacia G4 was unable to degrade TCE when grown without an inducer. Pseudomonas cepacia 5223-PR1, did display the ability to degrade TCE. However, the possible successful degradation of TCE in Shields et al relies on recombinant microorganisms.
Nelson et al in "Trichloroethylene Metabolism by Microorganisms That Degrade Aromatic Compounds", Applied and Environmental Microbiology, Vol. 54, pg. 604-606, (February 1988), tested six strains known for degrading naphthalene, biphenyl and toluene for potential ability to degrade TCE. Pseudomonas putida PpF1 and Pseudomonas putida B5 demonstrated some ability to degrade TCE. However, successful TCE degradation occurred only upon induction by addition of toluene or phenol. Of course, it is critically disadvantageous in remediating contaminated soils, ground water and waste materials when induction or degradation requires addition of further toxic substances to the media subjected to the remediation treatment.
Whited et al in "Toluene-4-Monooxygenase, a Three-Component Enzyme System That Catalyzes the Oxidation of Toluene to p-Cresol in Pseudomonas mendocina KR1", Journal of Bacteriology, Vol. 179, p.3010-3016, (May 1991), discloses oxidation of toluene to p-cresol, the degradation being initiated by toluene-4-monooxygenase enzyme. No ability to degrade TCE was tested or found.
Subsequent to Whited et al, Yen et al in "Cloning and Characterization of a Pseudomonas mendocina KR1 Gene Cluster Encoding Toluene-4-Monooxygenase", Journal of Bacteriology, Vol. 173, p. 5315-5327 (September 1991), further investigated the toluene-4-monooxygenase pathway for hydroxylation of toluene to p-cresol. Although they acknowledge the importance of T4MO in the degradation of trichloroethylene, they did not perform any testing in this regard. Moreover, they employed recombinant Escherichia coli.
U.S. Pat. No. 5,079,166 discloses a method of microbial degradation of trichloroethylene employing Pseudomonas mendocina KR-1. However, '166 employs recombinant cells treated with an inducer of toluene monooxygenase (TMO) genes. '166 further discloses the degradation of TCE with recombinant Pseudomonas putida Y2101 cells. The recombinant Y2101 cells are also treated with an inducer of TMO. Certain of the cell lines from Pseudomonas mendocina KR-1 employ particular inducers. For example, pKY277, pKY280, pKY281 and pKY282 employ toluene as an inducer. Similarly, pMY402, pMY405, pMY401 and pMY404 employ isopropyl-.beta.-D-thiogalactopyranoside. pKY287 employs a change in temperature as the inducer. Of course, '166 has the disadvantage in the remediation context of utilizing recombinant organisms and requiring an inducer to initiate degradation.
Winter et al, "Efficient Degradation of Trichloroethylene By a Recombinant Escherichia coli" (Bio/Technology, pp 282-285, March 1989), discloses much of the subject matter as the '166 patent, including duplicative cell lines.
All of the above examples have the disadvantages of either not working to successfully degrade VOC's, require toxic or additional inducer substrates to achieve degradation of VOC, or rely on recombinant cell lines.
Many VOC's, especially TCE and its toxic properties, make it imperative that the contaminant be promptly, efficiently and effectively removed from locations where contact with humans is possible. Many constraints, however, frequently inhibit or prevent removal of the offending TCE. For example, in many instances the contaminant has progressed downwardly into the ground to a point where excavation is impossible or impractical. The soil must therefore be decontaminated in situ. This critical common restriction eliminates the potential use of many possible decontamination technologies, such as those described above. Many alternative technologies have been attempted in efforts to effectively and efficiently decontaminate TCE polluted areas. However, these technologies have proven inadequate in achieving complete remediation or are environmentally incompatible, practically not viable or cost prohibitive. For example, a number of technologies treat groundwater or soil, but not both, which is highly desirable to achieve complete remediation in many cases. Similarly, the need for toxic inducers in several technologies is quite impractical.